Compositions and methods for inhibiting cellular adhesion or directing diagnostic or therapeutic agents to RGD binding sites

ABSTRACT

Compounds comprising R-G-Cysteic Acid (i.e., R-G-NH—CH(CH 2 —SO 3 H)COOH or Arg-Gly-NH—CH(CH 2 —SO 3 H)COOH) and derivatives thereof, including pharmaceutically acceptable salts, hydrates, stereoisomers, multimers, cyclic forms, linear forms, drug-conjugates, pro-drugs and their derivatives. Also disclosed are methods for making and using such compounds including methods for inhibiting cellular adhesion to RGD binding sites or delivering other diagnostic or therapeutic agents to RGD binding sites in human or animal subjects.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/943,900 filed Nov. 10, 2010 and issuing on Apr. 28, 2015 as U.S. Pat.No. 9,018,352, which claims priority under 35 U.S.C. 119(e) to U.S.Provisional Patent Application Ser. No. 61/259,748 entitled CompositionsAnd Methods For Inhibiting Cellular Adhesion To RGD Binding Sites filedon Nov. 10, 2009, the entire disclosure of each such prior patent andapplication being expressly incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the fields of chemistry andmedicine and more particularly to compositions of matter and methodsuseable for inhibiting cellular adhesion to Arg-Gly-Asp (the “RGD”tripeptide) binding sites and related treatments for disorders such asinflammation, wound-healing, prevention of scar formation, thrombosis,cancer metastasis and tumors, proliferative or non-proliferativediabetic retinopathy, liquefaction of the vitreous humor, induction ofposterior vitreo-retinal detachment (PVD), pathogenesis of vitreoretinaldiseases, such as floaters, idiopathic macular hole, vitreomaculartraction, age related macular degeneration, wet macular degeneration,choroidal neovascularization, vitreoretinal surgery, vein occlusion,corneal neovascularization, ischemic optic nerve, rubiosis iridis andprevention of scar formation in glaucoma surgery.

BACKGROUND OF THE INVENTION

The RGD tripeptide sequence is found in a number of proteins, where itplays a role in cell adhesion. Examples of proteins in which the RGDtripeptide sequence is present include collagens, fibronectin,vitronectin, von Willebrand factor (VWF), certain disintegrins, andcertain discoidins.

Integrins are heterodimeric cell surface receptors which mediateadhesion between cells and the extracellular matrix (ECM) by binding toligands having an exposed RGD sequence. Normal integrin-RGD binding isbelieved to play a role in gene expression involved in cell growth,migration, and survival. Faulty regulation of such cell growth,migration, and survival can result in a number of disease statesincluding thrombosis, inflammation, and cancer. Thus, RGD peptides havebeen investigated as potential mimics of cell adhesion proteins and fortheir ability to bind to integrins, for therapeutic purposes such asinhibiting apoptosis, angiogenesis, tumorigenesis, for the use in theirmultimeric form as internal radiotherapeutic agents as well as cancerimaging agents and for their anti-cancer drug carrying abilities.

In the eye, integrins affect a number of processes including oculardevelopment, cell migration, healing and some pathologic processes.Integrins may also modulate inflammation and thrombosis in oculartissue. Intravitreally injected RGD peptide has also been reported tocause posterior vitreoretinal detachment in an animal model and, thus,may be useful in the treatment of certain retinal disorders and/or tofacilitate removal of the vitreous body in a vitrectomy procedure. [SeeOlivera, L. B., et al., RGD Peptide-Assisted Vitrectomy to FacilitateInduction of a Posterior Vitreous Detachment: a New Principle inPharmacological Vitreolysis; Current Eye Research (8):333-40 (Dec. 25,2002)].

SUMMARY OF THE INVENTION

The present invention provides novel compounds comprising R-G-CysteicAcid (i.e., R-G-NH—CH(CH₂—SO₃H)COOH or Arg-Gly-NH—CH(CH₂—SO₃H)COOH) andderivatives thereof (including pharmaceutically acceptable salts,hydrates, stereoisomers, multimers, cyclic forms, linear forms,drug-conjugates, pro-drugs and their derivatives).

The present invention also provides compositions and methods forinhibiting cellular adhesion to RGD binding sites or delivering otherdiagnostic or therapeutic agents to RGD binding sites in human or animalsubjects by administering to the subject an effective amount of acomposition comprising an R-G-Cysteic Acid peptide or a derivativethereof (including pharmaceutically acceptable salts, hydrates,stereoisomers, multimers, cyclic forms, linear forms, drug-conjugates,pro-drugs and their derivatives).

Specific examples of R-G-Cysteic Acid peptide of this invention includea linear form of Arg-Gly-NH—CH(CH₂—SO₃H)COOH (example referred to hereinas Compound 1) and a cyclic form of Arg-Gly-NH—CH(CH₂—SO₃H)COOH)(example referred to herein as Compound 2).

General formulas for R-G-Cysteic Acid derivatives of the presentinvention include but are not limited to compounds having GeneralFormulas I-VII as follows:

where X is selected from: H, C₁-C₆ alkyl, Ph or SO₃H and Y═OH or NH₂.

where X is selected from: H, C₁-C₆ alkyl, Ph or SO₃H.

where X is selected from: H, C₁-C₆ alkyl, Ph or SO₃H and wherein Z isselected from: H or SO₃H

where X is selected from: H, C₁-C₆ alkyl, Ph or SO₃H; Y is selected fromOH or NH₂.

where X is selected from: H. C₁-C₆ alkyl, Ph or SO₃H.

where X is selected from: H, C₁-C₆ alkyl, Ph or SO₃H.X₁—R-G-Cysteic Acid-X  General Formula VII:where X and X₁ are selected from: cyclic or linear -Phe-Val-Ala,-Phe-Leu-Ala, -Phe-Val-Gly, -Phe-Leu-Gly, -Phe-Pro-Gly, -Phe-Pro-Ala,-Phe-Val, or any salt of any combination of the D-isomer or L-isomer of:Arg, Gly, Cysteic, Phe, Val, Ala, Leu, Pro, Thr.

Examples of cyclic forms of General Formula VII include but are notnecessarily limited to:

where X′ is selected from: H, C₁-C₆ alkyl, Ph or SO₃H and Z is selectedfrom H or Me; Y is selected from OH, NH₂.

Sulfonic acids are stronger acids than corresponding carboxylic acids.This higher polarity of the sulfonic acid group leads to strongerintermolecular bonding. For example, R-G-Cysteic acid, which has a morepolarized O—H bond, may form stronger hydrogen bonds than R-G-Asparticacid (RGD peptide), which has a relatively less polarized O—H bond, withthe amide groups of the proteins in the integrin binding site and/orhave stronger interactions with metal ions complexed in the integrinbinding site.

As described in more detail elsewhere herein, one specific example ofGeneral Formula VII,Glycinyl-Arginyl-Glycinyl-Cysteic-Threonyl-Proline-COOH (GRG CysteicAcid TP; referred to below as Compound 1) was synthesized and tested inanimals and found to be effective in inducing posterior vitreousdetachment (PVD) from retina surface by inhibitingintegrin-extracellular matrix (ECM) interactions. As described also inmore detail elsewhere herein, Compound 1 was tested in a model of woundhealing using human umbilical vein endothelial cells (HUVEC) and wasshown to inhibit cell adhesion by 74% in 12 hours compared to aninhibition of 40% by a cyclic-RGD peptide. These studies suggest andcorroborate the rationale thatGlycinyl-Arginyl-Glycinyl-Cysteic-Threonyl-Proline-COOH may bind tointegrin even more strongly than RGD peptides themselves.

The RGCysteic Acid peptide sequence, which can be in either L-form orD-form, is a competitive inhibitor of integrin-ECM interactions. TheRGCysteic Acid peptide sequence can be of protease-resistant derivativesor of cyclic derivatives or of pro-drug derivatives or associated withdrug delivery systems or of monoclonal antibodies.

The compositions of the present invention are useable to inhibitangiogenesis, which can be useful for treating inflammation,wound-healing, thrombosis, cancer metastases and tumors. Further usefulapplication can be found in ophthalmology including, proliferative ornon-proliferative diabetic retinopathy, liquefaction of the vitreous,induction of posterior vitreo-retinal detachment (PVD), pathogenesis ofvitreoretinal diseases, such as idiopathic macular hole, vitreomaculartraction, age related macular degeneration, wet macular degeneration,choroidal neovascularization, vitreoretinal surgery, vein occlusion, andprevention of scar formation in glaucoma surgery. Still further,multimeric and/or radiolabeled compositions of the present invention areuseable as diagnostic/imaging agents for the detection of tumors andradiotherapeutic agents for the treatment of tumors and as anti-cancerdrug carriers due to their tumor directing properties.

Biomaterials incorporating an RGC peptide can also provide a syntheticadhesive microenvironment for the long term survival and growth of cellsand for the engineering of living tissues for applications in tissueengineering and regenerative medicine. Through their property of bindingintegrin adhesion receptors, RGC peptides can provide anadhesion-promoting signal when it is tethered onto a biomaterial orscaffold. RGC-based materials mediate cell adhesion, spreading, andmigration of cells. In addition, integrin-mediated cell adhesionpromotes cell proliferation and survival and plays a key role inassembly and organization of multicellular structures and tissues.

Drugs for the treatment of macular degeneration like Lucentis andAvastin are based on inhibiting VEGF, which otherwise causes the growthof new vessels, angiogenesis, and consequently contributes macularedema. It has been known that a small peptide RGD can induce apoptosisby inhibiting cell attachment to extracellular matrix¹ by competitivebinding as shown in U.S. Pat. No. 6,500,924 to Brooks et al.

The RGD peptide binding or recognition motif can be found in theproteins of extracellular matrix and integrins which link theintracellular cytoskeleton of cells with the ECM by recognizing the RGDadhesion epitopes. See, for example, Foos, R Y., Invest. Opthalmol. Vis.Sci. (1972) 11, 801-808. Cells, without the attachment to the ECM,normally undergo apoptosis.

In general, interactions between fibroblasts and glycoprotein componentsof extracellular matrix cause a major scar formation mediated primarilyby the RGD containing amino acid sequence interacting on the cellsurface integrins. It has also been known that the RGD sequence isinvolved in cell-ECM interactions during inflammatory and homeostaticreactions (see Hershkoviz, S. M., et al., Invest. Ophthalmol. Vis. Sci.,(1994), 35, 2585-2591) and the integrins play an important role in cellmigration in wound healing or pathologic processes and modulatinginflammation and thrombosis. Thus, potent integrin antagonists, like RGDpeptides, might be very useful as pharmacologic agent asanti-inflammatory, anti-metastatic or anti-thrombotic agents (see Elner,S. G. and Elner, V. M., IOVS (1996) 37:696-701. It is also reported inliterature that CD44 receptor for hyaluronic acid mediates cell-cell andcell-matrix interactions through its affinity for hyaluronic acid, andpossibly also through its affinity for other ligands such asosteopontin, collagens, and matrix metalloproteases (MMPs).

Adhesion with hyaluronan plays an important role in cell migration,tumor growth and progression and also involved in lymphocyte activation,recirculation and homing, and hematopoiesis. Altered expression ordysfunction causes numerous pathogenic phenotypes (see for example,Jiang D., Annu. Rev. Cell. Dev. Biol. (2007) 23: 435-461; and Knudson,W. et al, Matrix Bio. (2002), 21: 15-23).

Recently, it has been shown that the interaction of CD44 and cell-matrixcomponents (e.g., HA) plays a significant role in the development ofvarious inflammatory diseases and interruption of hyaluronan-CD44interactions would lead to amelioration of choroidal neovascularization(see Hiroshi Mochimaru et al., Invest. Ophthalmol. Vis. Sci. (2009) 50:4410-4415).

These evidences demonstrate that an adhesion molecule like RGD peptideor CD44 in cell-cell and cell-ECM interactions plays an important rolein the development of numerous pathogenic diseases and the inhibition ofthe interactions can be a novel therapeutic target in treating andcuring the diseases.

Synthetic peptides have also been shown to bind to integrins and growthfactors. Cyclized pentapeptides containing RGD sequences have been foundto inhibit binding of vitronectin to α_(v)β₃ integrin (see Michael A.Dechantsreiter, et al., J. Med. Chem. (1999) 42:3033-3040) and bothvitronectin and fibronectin to α_(v)β₃ and α_(llb)β₃ integrins (seeRoland Haubner et al., J. Am. Chem. Soc., (1996)118:7461-7472). Thisinhibition has been shown to be useful in the treatment of multiple,unrelated diseases. In hamster studies, the cyclic pentapeptides delayedgrowth and metathesis of tumors in comparison with control animals (seeM. A. Buerkle et al., British J. Cancer (2002) 86: 788-795). Thepentapeptides have also been shown to reduce binding of sickle red bloodcells to vascular endothelium and improved hemodynamic behavior (seeEileen M. Finnegan et al., Am. J. Physiol. Heart Circ. Physiol., (2007)293: H1038-H1045). Another cyclic peptide containing the RGD sequencehas shown strong binding to α4β1, an integrin known to play a role inleucocyte binding in inflammatory and immune responses (see Pina M.Cardarelli et al., J. Biol. Chem. (1994) 269:18668-18673). A synthetic,sulfated tetrapeptide has been shown to strongly bind to VEGF (seeMaynard, J. A. Hubbell, Acta Biomaterialia (2005) 1: 451-459).

In addition, in an important and useful application, a dimeric RGDpeptide-drug conjugate has been shown to be useful for integrin-targeteddrug delivery for tumor targeting (see Chen et al., J. Med. Chem.,(2005) 48(4): 1098-1106).

In another equally important and useful application, multimericradiolabeled RGD peptides have been shown to be useful asdiagnostic/imaging agents for tumor detection and, as radiotherapeuticagents for tumor specific targeting and treatment by targeting theintegrin α_(v)β₃ (see Zi-Bo Li et al., J. Nucl. Medicine, (2007) 48:1162-1171).

In ophthalmology, scar formation in wound healing by fibroblast is oneof the major problems, particularly in glaucoma. This arises frominteractions between fibroblast and glycoprotein components of ECM.Recognition of ECM glycoproteins occurs via cell surface integrins thatare specific for adhesion epitopes, such as the Arg-Gly-Asp (or RGD)sequence. The RGD sequence, which is present in several matrices ofplasma proteins, including fibronectin (FN) and vitronectin (VN), isinvolved in cell-ECM interactions that occur during inflammatory andhomeostatic reactions. Inhibition of the interactions between fibroblastand glycoproteins of ECM alleviated the scar formation (see RamiHershkoviz et al., Invest. Ophthalmol. Vis. Sci. (1994) 35: 2585-2591).

The collagen fibrils of the posterior vitreous cortex adhere to thevitreoretinal interface, specifically to the inner limiting lamina ofthe retina surface (see Sebag J., Eye (1992), 6: 541-552). The adherenceat the vitreous base, the optic disc, along the major retinal vesselsand in a facial manner to the entire posterior pole plays an importantrole in the pathogenesis of vitreoretinal diseases, such as idiopathicmacular hole, vitreomacular traction, proliferative diabeticretinopathy, etc. (Akiba J. Quiroz M. A., et al., Ophthalmol. (1990) 97,1619-1655).

Angiogenesis inhibition is showing early promise with diabeticretinopathy and macular degeneration, which both result from anovergrowth of ocular blood vessels. In these disorders, the vesselsinterfere with normal structures in the eye, or they block light fromreaching the back of the eye. The new blood vessels are themselves theprimary pathology, and stopping blood vessel growth could preventblindness. Thus, angioinhibition could result in not just a treatment ofthese disorders; it could be a cure. Furthermore, it has been postulatedthat separation of the vitreous from the retina can alleviate maculartraction, reducing the risk of macular hole formation. Accordingly, theposterior vitreous detachment by the inhibition of fibronectin andlaminin binding to the integrins on the vitreoretinal interface, mayprevent retinal neovascularization in eyes with diabetic retinopathy andretinal vein occlusion (see Akiba J. Quiroz M A, Ophthalmol. (1990) 97,1619-1655; Kelly N. E., et al., Arch. Ophthalmol. (1991) 109, 654-659;and Kado M. et al., Am. J. Ophthalmol. (1988) 105: 20-24).

In recent years, vitreous surgical procedures have been greatly improvedto relieve vitreoretinal tractions and to reduce retinal edema. Despitecontinued improvement in surgical techniques and instrumentation, itstill remains difficult to achieve an atraumatic removal of the vitreouscortex in some patients particularly in diabetic retinophathy andpediatric patients due to complications such as retinal breaks, retinaldetachment and retinal nerve fiber damage (see Sebag J., Arch. Clin.Exp. Ophthalmol. (1987) 225: 89-93; and Han D. P., et al., Arch.Ophthalmol. (1998) 106: 998-1000) etc. Thus, a less traumatic approachto selectively cleave the vitreous interface without damaging the retinais highly desirable. In recent years, reports concerning a number ofpharmacologic agents for the separation of the vitreoretinal interfacehave appeared in literature (see Trese M. T., Eye. (2002) 16: 365-368;Gandorfer A., et al., Am. J. Ophthalmol. (2002) 133: 156-159; and HesseL., et al., Eye Res. (2000) 70: 31-39). The pharmacologic vitreolysisusing enzymes such as hyaluronidase (see U.S. Pat. No. 6,863,886 toKarageozian et al.) and autologous plasmin (Sakuma T, et al., NipponGanka Gakkai Zassi (2003) 107: 709-718) have been explored to promotethe digestion of extracellular matrix and to induce posterior vitreousdetachment in the past. Yet, a non-specific destruction of adjacenttissues by the enzymes employed impedes success of their therapeuticapplication. In the last few years, a novel approach using non-enzymaticpharmacologic agents like urea (see Nickerson, C., et al., in J.Biomechanics, (2008) 41: 1840-1846, and in Macromol. Symp. (2005):183-189) and RGD peptide (see Leonardo B. Oliviera, et al., Curr. EyeRes. (2002) 25: 333-340) has been investigated by concentrating on theseparation of vitreoretinal interface. It has been shown that asynthetic analog of RGD peptide competes for the RGD motif of ECMproteins to disrupt integrin—ECM interactions and to loosen theattachments in-vitro (Williams J. A., Pathol. Bio. (1992) 40: 813-821;Gehlsen K. R., et al., J. Cell. Biol. (1988) 106: 925-930;Pierschbacher, M. D., et al., J. Biol. CHem., (1987) 262: 17294-17298;and Zhon L. L., et al., IOVS. (1996) 37: 104-113) and in-vivo. Thus, theintravitreal injection of soluble RGD peptides led to a release ofRGD-epitopes from the insoluble ECM proteins of the retinal surface,consequently facilitating the non-enzymatic PVD in rabbit models.Clearly, these results indicate that the vitreoretinal interfaceinvolves integrin connection to RGD motif of ECM as well as adhesion ofvitreous cortical collagens to the inner limiting lamella (ILL). RGDpeptides and their derivatives promote migration of epithelial cells ina wound (see P. M. Mertz el al., J. Burn Care Res. (1996) 17: 199-206)and maintain their bioactivity when incorporated into syntheticbiomaterials such as hydrogels (see M P Lutolf, et al., Proc. Nat. Acad.Sci. (2003) 100: 5413-5418; and M P Lutolf, et al., Nature Biotechnol.(2003) 21: 513-518), other polymer matrices (see Homg-Ban Lin et al., J.Biomed. Material. Res. (2004) 28: 329-342) or as surface films on hardsubstrates (D. M. Ferris et al., Biomaterials (1999) 20: 2323-2331). RGDpeptides also promote increased adhesion of epithelial or endothelialcells to vascular prostheses (see K. Walluscheck el al., Eur. J.Vascular and Endovascular Surgery (1996) 12: 321-330) and otherartificial organs (see Jeschke, Brigette, Biomaterials (2002) 23:3455-3463) coated with the peptide sequence and have been shown tosupport nerve regrowth (see M. Rafiuddin Ahmed et al., Brain Res. (2003)993:208-216). The prostheses's biologically active surface can containsynthetic resin fibers or polymers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of three peptides, namely, cyclic-RGD-peptide,RGC peptide (Compound 1) and RGE peptide, on the kinetics of woundhealing;

FIG. 2 shows and HPLC chromatogram of RGC peptide (Compound 1);

FIG. 3 shows an Electrospray mass chromatogram of RGC peptide (Compound1);

FIG. 4 shows and HPLC chromatogram of cyclic-RGC peptide (Compound 2);and

FIG. 5 shows an Electrospray mass chromatogram of cyclic-RGC peptide(Compound 2).

DETAILED DESCRIPTION AND EXAMPLES

The present invention provides novel compounds, including those ofGeneral Formulas I through VII above. Specific examples include linearform of Arg-Gly-NH—CH(CH₂—SO₃H)COOH (example referred to herein asCompound 1) and cyclic form of Arg-Gly-NH—CH(CH₂—SO₃H)COOH) (examplereferred to herein as Compound 2) as well as derivatives thereof,including pharmaceutically acceptable salts, hydrates, stereoisomers,mutimers, cyclic forms, linear forms, multimeric forms, drug conjugates,pro-drugs and their derivatives.

Synthesis of Compounds 1 and 2

Conventional solid-phase peptide synthesis (SPPS; see R. B. Merrifield,J. Am. Chem. Soc. (1963) 85 (14): 2149-2154) known to a person ofordinary skill in the art maybe carried out. The SPPS is a preferredmethod of synthesis because of the high yields. In general, the firststage of the solid phase peptide synthesis technique consists of peptidechain assembly with protected amino acid derivatives on a polymericsupport. The second stage of the technique is the cleavage of thepeptide from the resin support with the concurrent cleavage of all sidechain protecting groups to give the crude free peptide. The generalprinciple of SPPS is one of repeated cycles of coupling-deprotection.The free N-terminal amine of a solid-phase attached peptide is coupledto a single N-protected amino acid unit. This unit is then deprotected,revealing a new N-terminal amine to which a further amino acid may beattached. See Asymmetric Synthesis by Von G. M. Coppola and H. F.Schuster; John Wiley & Sons, New York 1987 for synthesis, protection anddeprotection strategies and Greene's Protective Groups in OrganicSynthesis by Peter G. M. Wuts and Theodora W. Greene, (2nd edition) J.Wiley & Sons, 1991. for protection and deprotection strategies.

Of the two major used forms of solid phase peptide synthesis—Fmoc(9-fluorenylmethyloxycarbonyl; base labile alpha-amino protecting group)and t-Boc (t-butyloxycarbonyl; acid labile protecting group), Fmoc maybe used preferably in the synthesis of the present peptides. Each methodinvolves different resins and amino acid side-chain protection andconsequent cleavage/deprotection steps. After cleavage from the resin,peptides are usually purified by reverse phase HPLC using columns suchas C-18, C-8, and C-4.

An Example of Solid Phase Peptide Synthesis

The following is an outline of the synthetic steps for peptide synthesison Wang resin as the solid support, using the base labile9-fluorenylmethyloxycarbonyl (Fmoc) protecting group.

Fmoc Deprotection

Load 0.08 mmol of Fmoc-Pro-Wang resin into a fritted column equippedwith a plastic cap. Wash the resin with 2×3-ml portions of DMF(dimethylformamide) for 1 minute each. Next, add about 3 ml of 20%piperidine in DMF and allow the Fmoc deprotection to continue for 15minutes. During this time, gently swirl or agitate the column to assurea complete mixing. After the reaction is complete (about 15 min.), drainthe reaction column and wash the resin again with DMF (4×3 ml).

Amide Bond Coupling

The desired Fmoc-protected amino acid, Fmoc-Thr-tBu, (3 eq.; relative toresin loading indicated by supplier) and DIEA (6 eq.) in DCM (0.5 M withrespect to the amino acid) are then added to the resin. The mixture iscooled at −20° C. for 20 minutes. Next,benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), a peptide coupling reagent used in solid phase peptidesynthesis (3 eq.) is added to the reaction. After shaking at −20° C. for8 hours, the reaction mixture is drained and the resin is washed withDCM (3×).

After the Fmoc deprotection using 20% piperdine in DMF (15 min) and thewash with DMF (3×), the next Fmoc protected amino acid (3 eq.; relativeto resin loading), PyBop (is coupled in the same manner as above.

Cleavage

In order obtain the peptide in the free acid form, the ester linkage iscleaved using strongly acidic conditions such as TFA (trifluoroaceticacid). Treat the resin with 2-3 ml of a solution of trifluoroacetic acidand water 95:5. Gently agitate the resin over a period of 25 minutes.Next, drain the column and carefully collect the filtrate into a glasscollection vessel.

Synthesis of Compound 1 (GRG Cysteic Acid TP):

Step 1. Resin Loading: An o-chlorotrityl resin preloaded with proline isused as the starting material.Step 2. Peptide assembly: Fmoc synthesis is used to assemble thepeptide. Protected amino acids are activated with PyBOP and the terminalFmoc groups removed with 20% piperidine in DMF. The following protectedamino acids are used in the order in the order in which they appear:

a. Fmoc-Thr-tBu (Fmoc threonine-t-butyl ester)

b. Fmoc-cysteic acid-Pfp (Fmoc cysteic acid-pentafluorophenyl ester)

c. Fmoc-Gly (Fmoc glycine)

d. Fmoc-Arg-Pbf (N_(α)-Fmoc-N_(ω)-(2,2,4,6,7pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine)

e. Fmoc-Gly (Fmoc glycine)

Step 3. Peptide cleavage from Resin: The resulting peptide is cleavedfrom the solid support and the protecting groups removed with a solutionof 85.5% TFA, 5% phenol, 2% water, 5% thioanisole, and 2.5%ethanedithiol.

Step 4. Purification: High Performance Liquid Chromatography (HPLC), isused to purify the resulting RGC peptides.

A quantity of Compound 1, prepared as described above, was analyzed byHigh Performance Liquid Chromatography to be >98% area/area in purity[HPLC conditions: Buffer A: 0.1% trifluoroacetic acid (TFA) in water,Buffer B:0.1% TFA in acetonitrile, Mobile Phase(MP) A: 97% Buffer A and3% Buffer B, Mobile Phase B: 79% Buffer A and 21% Buffer B. Mobile PhaseC: 50% Buffer A and 50% Buffer B; for gradient see Table 1 below; flowrate: 1.0 mL/minute; column: Waters Symmetry® C18, 5μ, 4.6×250 mm;column temperature: 30° C.; detector: UV@220 nm; sample injectionvolume: 20.0 μL; sample preparation: 20 μL sample diluted with 1.0 mLMobile Phase A (approximately 0.5 mg/mL)]. The corresponding HPLCchromatogram is shown in FIG. 2. In addition, based on the stepwiseaddition of the corresponding amino acids for the synthesis of thepeptide sequence, the molecular weight of purified Compound 1 wasdetermined by Electrospray Mass Spectrometry to be 638.3 amu(theoretical mass: 637.7 amu), confirming the identity of Compound 1.The Electrospray mass spectrogram of Compound 1 is shown in FIG. 3.

TABLE 1 Pump Gradient Program to detect Compound 1 by HPLC Pump GradientProgram Step Time % MP A % MP B % MP C 0 0.1 min  100 0 0 1 60 min 0 1000 2 20 min 0 0 100 3 15 min 100 0 0Synthesis of Compound 2 (Cyclo-RGCysteic Acid fN(CH₃)V):

Step 1. Loading Resin: An o-chlorotrityl resin is used as the startingmaterial. Fmoc-N□-methyl-L-Val is attached to the resin.Step 2. Peptide Assembly: 2. Fmoc synthesis is used for the peptideassembly. Protected amino acids are activated with PyBOP and theterminal Fmoc groups removed with 20% piperidine in DMF. The followingprotected amino acids are used in the order in which they appear:

a. Fmoc-Phe (Fmoc-phenyl alanine)

b. Fmoc-cysteic acid-PfP (Fmoc-cysteic acid pentafluorophenyl ester)

c. Fmoc-Gly (Fmoc-glycine)

d. Fmoc-Arg-Pbf (N_(α)-Fmoc-N_(ω)-(2,2,4,6,7pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine)

Step 3. Peptide cleavage from resin: The peptide is cleaved from thesolid support using acetic acid/TFA/DCM (1:3:3)

Step 4. Cyclization and deprotection to desired cyclic peptide:

Cyclization via in situ activation using diphenylphosphorylazide andsodium bicarbonate under high dilution. The side chains are deprotectedusing 85.5% TFA, 5% phenol, 2% water, 5% thioanisole, 2.5%ethanedithiol.

Step 5. Purification: HPLC is used for purification.

A quantity of Compound 2, prepared as described above was analyzed byHigh Performance Liquid Chromatography to be >99% area/area in purity(HPLC conditions: Mobile Phase A: 0.1% trifluoroacetic acid in water, B:0.1% TFA in (80% acetonitrile plus 20% water); gradient 26% to 36% B in20 minutes; flow rate: 1.0 mL/minute; column:Phenomenex C18(2) 4.6×150mm, 5μ, 100 A; detector: UV@220 nm; sample injection volume: 100.0 μL).The corresponding HPLC chromatogram is shown in FIG. 4. In addition,based on the stepwise addition of the corresponding amino acids for thesynthesis of the peptide sequence, the molecular weight of purifiedCompound 2 was determined by Electrospray Mass Spectrometry to be 625.3amu (theoretical mass: 625.77 amu), confirming the identity of Compound2. The Electrospray mass spectrogram of Compound 2 is shown in FIG. 5.

Methods for Inhibiting Cellular Adhesion

The present invention also provides methods for inhibiting cellularadhesion to RGD binding sites in a human or animal subject byadministering to the subject an effective amount of R-G-Cysteic Acid(i.e., linear form of R-G-NH—CH(CH₂—SO₃H)COOH or cyclic form ofR-G-NH—CH(CH₂—SO₃H)COOH) or a derivative thereof (includingpharmaceutically acceptable salts, hydrates, stereoisomers, multimers,cyclic forms, linear forms, drug-conjugates, pro-drugs and theirderivatives).

Applicant has discovered that synthetic RGCysteic Acid peptides of thepresent invention induce apoptosis by competitively inhibiting the cellattachment to the ECM components. Therefore, the present syntheticRGCysteic acid peptides and their derivatives can be used as potentintegrin antagonists as therapeutic agent against angiogenesis,inflammation, cancer metastasis, thrombosis, prevention and treatment ofscar formation as well as a pharmacological vitreolysis agents. Inaddition, in an important aspect of the present invention, an improvedtargeting of the α, β integrins using multimerized and radiolabeled RGCpeptides, for use as internal radiotherapeutic agents for tumordetection (diagnostic or imaging agent) and tumor treatment isenvisaged. In another important aspect, improved RGC peptide conjugatesor multimeric RGC peptide conjugates function as drug carriers, forexample, as anti-cancer drug carriers for efficient tumor targeting.

As described elsewhere herein, the sulfonic acids in the RGC-peptidesare stronger acids than corresponding carboxylic acids in RDG-peptides.This higher polarity of the sulfonic acid group leads to strongerintermolecular bonding. For example, R-G-Cysteic acid, which has a morepolarized O—H bond, may form stronger hydrogen bonds than R-G-Asparticacid, which has a relatively less polarized O—H bond, with the amidegroups and/or side chains of the amino acids of the proteins in theintegrin binding site and/or have stronger interactions with metal ionscomplexed in the integrin binding site.

Therefore, the novel RGC peptides and their derivatives of the presentinvention present improved compounds and compositions over thecorresponding RGD peptides in integrin receptor recognition and binding.

Additionally, in diabetic patients with chronic hyperglycemia associatedwith elevated IOP, open angle glaucoma particularly is reported to berelated to the accumulation of fibronectin in the trabecular meshworktissue and the excess of fibronectin is believed to inhibit the aqueousoutflow (see Oshitari, T, et al., Am. J. Ophthalmol. (2007)143:363-365). Involvement of fibronectin in the cell-ECM interaction inthe trabecular meshwork was indicated in primary open angle glaucoma(see Mark S. Filla, et al., Invest. Ophthalmol. Vis. Sci. (2002)43:151-161; and Cheryl R. Hann, et al., Ophthalmic Res. (2001) 33:314-324). It has also been reported that the endothelial cells ofSchlemm's canal interact with extracellular matrix in influencingoutflow facility (see Cindy K. Bahler et al., Invest. Ophthalmol. Vis.Sci. (2004) 45: 2246-2254). In light of the inhibition of the aqueousoutflow by the presence of an excess of extracellular matrix componentssuch as fibronectin, application of RGCysteic acid peptides and theirderivatives to treat the elevated IOP of diabetic patients would behighly beneficial to diabetic patients.

The preferred RGCysteic acid peptides can be a fusion polypeptide, acyclic or linear polypeptide, a derivatized polypeptide, includingRGCysteic acid peptide derivatized or associated or coupled with drugdelivery systems or other drugs, such as, for example, anti-cancerdrugs, a multimerized RGC peptide, a monoclonal antibody containingRGCysteic acid sequence that immunoreacts with integrins' binding siteor functional fragment thereof.

RGCysteic acid containing polypeptides can have a sequence correspondingto the amino acid residue sequence of natural integrin adhesive bindingregion, such as those present in fibrinogen, fibronectin, vitronectin,von Willebrand factor, laminin, thrombospondin, and the like ligands.

The present peptide sequence consists of three amino acids havingterminal guanidino, sulphonic and carboxylic groups and its derivativescoupled and/or associated with drug delivery systems, including peptidefragments, glycoproteins, and polymer groups such as PEG, Pluronic, andother polymer groups, and, liposomes and nanoparticles. Pharmaceuticalcompositions comprising them for treatment of various pathologicaldisorders include injectable, gel, suspension, ointment, solid andliquid dosage forms.

Integrin receptors associated with a cell adhesion motif such asfibronectin, vitronectin, laminin, fibrinogen, thrombospondin, and vonWillebrand factor, are the target epitope of RGCyteic acid peptide andits derivatives. The tripeptide, RGCysteic acid, has been discovered asa recognizable minimal amino acid sequence by cell binding domains. Thissequence can also interfere with immune functions unrelated tointegrins.

Thus, it has been discovered that the synthetic RGCysteic acid sequenceis to mimic the RGD cell binding domain, and a substitution on theα-carbon of aspartic acid gives a stronger binding affinity to thetarget integrins. The sulfonic acids in the RGC-peptides are strongeracids than corresponding carboxylic acids in RDG-peptides. This higherpolarity of the sulfonic acid group leads to stronger intermolecularbonding. For example, R-G-Cysteic acid, which has a more polarized O—Hbond, may form stronger hydrogen bonds than R-G-Aspartic acid, which hasa relatively less polarized O—H bond, with the amide groups and/or sidechains of the amino acids in the integrin binding site and/or havestronger interactions with metal ions complexed in the integrin bindingsite.

The most general formulas for the RGCysteic acid sequences of thepresent invention are as follows:

Where X═

-   -   —CH(R₁)—S(═O)₂—Y;    -   —CH(R₁)—SH;    -   —CH(R₁)—OZ;    -   —CH(R₁)S(═O)Y;    -   —CH(R₁)—O—S(═O)₂—OX₁; and    -   —CH(R₁)—O—P(═O)—OX₁; and        Wherein Y═OX₁, NH₂; X₁═—H, C₁-C₆ straight chain alkyl, phenyl;        R₁═H, C₁-C₆ straight chain alkyl, phenyl or SO₃H        Z═H, SO₃H

Where X═

-   -   —CH(R₁)—S(═O)₂—Y;    -   —CH(R₁)—SH;    -   —CH(R₁)—OZ;    -   —CH(R₁)S(═O)Y;    -   —CH(R₁)—O—S(═O)₂—OX₁; and    -   —CH(R₁)—O—P(═O)—OX₁; and        Wherein Y═OX₁, NH₂; X₁═—H, C₁-C₆ straight chain alkyl, phenyl;        R₁═H, C₁-C₆ straight chain alkyl, phenyl or SO₃H        Z═H, SO₃H; and        A₂ is selected from: -Phe-Val-Ala, -Phe-Leu-Ala, -Phe-Val-Gly,        -Phe-Leu-Gly, -Phe-Pro-Gly, -Phe-Pro-Ala, -Phe-Val, or salt or        N-alkylated derivative thereof. Any combination of D-form or        L-form of Arg, Gly, Cysteic, Phe, Val, Ala, Leu, Pro, Thr, as        well as cyclic form of the above sequence can be used.        Examples of cyclic forms include:

where X′ is selected from: H, C₁-C₆ alkyl, Ph, SO₃H; Y═OH, NH₂ and Z═H,CH₃.

Cyclic forms also include penta and hepta peptides, such as for examplea specific compound of General Formula C, namely, Compound 2(Cyclo-RGCysteic Acid fN(CH₃)V), is shown below:

Formula A encompasses General Formulas I-VI described elsewhere herein.Formula B encompasses general formula VII described elsewhere herein.A₁-Arg-Gly-NH—CH(X)—CO-A₂  Formula D:Where X═

-   -   —CH(R₁)—S(═O)₂—Y;    -   —CH(R₁)—SH;    -   —CH(R₁)—OZ;    -   —CH(R₁)S(═O)Y;    -   —CH(R₁)—O—S(═O)₂—OX₁; and    -   —CH(R₁)—O—P(═O)₂—OX₁; and        Wherein Y═OX₁, NH₂; X₁═—H, C₁-C₆ straight chain alkyl, phenyl;        R₁═H, C₁-C₆ straight chain alkyl, phenyl or SO₃H        Z═H, SO₃H; and        A₁ and A₂ are selected from: -Phe-Val-Ala, -Phe-Leu-Ala,        -Phe-Val-Gly, -Phe-Leu-Gly, -Phe-Pro-Gly, -Phe-Pro-Ala,        -Phe-Val, or salt or N-alkylated derivative thereof. Any        combination of D-form or L-form of Arg, Gly, Cysteic, Phe, Val,        Ala, Leu, Pro, Thr, as well as cyclic form of the above sequence        can be used.

The substituted RGCysteic acid sequences includes cyclic RGCysteic acidanalogues.

The application of the RGCysteic acid and its derivatives can be madesubcutaneously, dermatologically, ophthamically and systemically, byemploying a drug delivery system or any pharmaceutically acceptabledosage forms of injection or solid or ointment formulation.

The compounds of the present invention may be administered by any routethat is suitable to bring about the intended therapeutic effectincluding but not limited to: oral, rectal, intravenous, intraarterial,intradermal, subcutaneous, intramuscular, intrathecal, sublingual,buccal, intranasal, transmucosal, transdermal, topical, intraocular,intravitreal, other enteral, other parenteral and/or other possibleroute(s) of administration.

The compounds of the present invention may be administered at any dosagethat provides the intended therapeutic effect while avoiding untoward ortoxic effects. Typical dosages at which the compounds of the presentinvention may be administered to human subjects are in the range ofabout 1 ng/kg to about 1.0 g/kg.

Where possible and appropriate, compounds of the present invention mayoptionally be prepared in the form of liposomes or nanoparticles (e.g.,nanocapsules). The formation and use of liposomes is generally known tothose of skill in the art. Liposomes are formed from phospholipidsdispersed in an aqueous medium such that they spontaneously formmultilamellar concentric bilayer vesicles sometimes referred to asmultilamellar vesicles (MLVs). MLVs are typically from 25 nm to 4 μm indiameter. When sonicated, MLVs form small unilamellar vesicles (SUVs) ofabout 200 to 500 angstroms in diameters having cores which contain theaqueous solution. In general, when dispersed in an aqueous medium,phospholipids can form various structures other than liposomes,depending on the molar ratio of lipid to water.

At low molar lipid to water ratios, liposomes will form. The physicalcharacteristics of liposomes depend on pH, tonicity and the presence ornon-presence of divalent cations. Liposomes can interact with cells bydifferent mechanisms, including 1) endocytosis (e.g., phagocytosis ofthe liposome by cells such as macrophages and neutrophils), adsorptionto the cell surface, 2) interaction with cell-surface components, 3)fusion with the plasma cell membrane by insertion of the lipid bilayerof the liposome into the plasma membrane or 4) transfer of liposomallipids to cellular or subcellular membranes, or vice versa. Varying theliposome formulation can alter which mechanism(s) by which the liposomeswill interact with cells in the paranasal sinus, nasal mucosa, etc.

A nanocapsule is any nanoparticle that consists of a shell and a space,in which desired substances may be placed. Techniques for formingnanocapsules are known in the art. Polymeric nanocapsules can be made inspecific sizes and shapes. They can be produced as monodisperseparticles which have precisely defined physical and chemical propertiesand, thus, can be tailored to facilitate release of the therapeutic ordiagnostic substance in response to particular bimolecular triggeringmechanisms, such as pH, mucous flow or other conditions present withinthe paranasal sinus or other area in the ear, nose or throat where thedevice is implanted. Nanocapsules can be used in the present inventionas “smart drugs” which have specific chemical receptors or binding sitesthat will bind to specific target cells (e.g., cancer cells or cellsassociated with inflammatory conditions.

The following are non-limiting examples of formulations forpharmaceutical preparations containing compounds of the presentinvention. Also included are examples of the safety and/or efficacydemonstrated using exemplary RGC peptides or derivatives in inhibitingcell adhesion. As used herein, the terms “RGCysteic Acid Peptide,” “RGCpeptide”, “RGCys-peptide” and “compounds of the present invention” shallsynonymously mean compositions containing the sequence R-G-Cysteic Acidand their derivatives, including but not limited to those defined byGeneral Formulas I-VII and Compounds 1, and 3-5 as described herein.

Example 1 Pharmaceutical Formulations

The following are examples of pharmaceutical formulations I-X, whichcontain R-G-Cysteic Acid Peptides of the present invention, such as anyof those defined by General Formulas I-VII or any of Compounds 1-5 asdescribed herein.

Formulation I R-G-Cysteic Acid Peptide (RGCys-peptide) 0.0001 mg to 10 gNaCl  0.01 mg to 0.9 g Water QS to 100.0 mL

Formulation II RGCysteic Acid Peptide (RGCys-peptide) 0.0001 mg to 10 gEDTA  0.001 mg to 100 mg NaCl  0.01 mg to 0.9 g Water QS to 100.0 mL

Formulation III RGCysteic-peptide 0.0001 mg to 10 g EDTA  0.001 mg to100 mg NaCl  0.01 mg to 0.9 g Citric Acid 0.0001 mg to 500 mg Water QSto 100.0 mL

Formulation IV RGCysteic-peptide 0.0001 mg to 10 g NaCl  0.01 mg to 0.9g Phosphate Buffer to pH = 3.0-9.0 Water QS to 100.0 mL

Formulation V RGCysteic-peptide 0.0001 mg to 10 g EDTA  0.001 mg to 100mg NaCl  0.01 mg to 0.9 g Phosphate Buffer to pH = 3.0-9.0 Water QS to100.0 mL

Formulation VI RGCysteic-peptide 0.0001 mg to 10 g NaCl  0.01 mg to 0.9g Borate Buffer to pH = 3.0-9.0 Water QS to 100.0 mL

Formulation VII RGCysteic-peptide 0.0001 mg to 10 g Hyaluronic AcidSodium salt  0.01 to 10% Boric Acid  0.01 to 1.0% Polyethyleneglycol(PEG 8000)  0.01 to 10% Sodium Chloride  0.10 to 0.9% Potassium Chloride 0.01 to 0.20% Calcium Chloride Dihydrate 0.001 to 0.05% MagnesiumChloride Hexahydrate  0.01 to 0.20% Preservative pH 4.0-8.0 Water QS to100.0 mL

Formulation VIII RGCysteic-peptide 0.0001 mg to 10 g Hyaluronic AcidSodium salt  0.01 to 10% Carboxymethyl Cellulose  0.01 t0 10% Boric Acid 0.01 to 1.0% Polyethyleneglycol (PEG 8000)  0.01 to 10% Sodium Chloride 0.10 to 0.9% Potassium Chloride  0.01 to 0.20% Calcium ChlorideDihydrate 0.001 to 0.05% Magnesium Chloride Hexahydrate  0.01 to 0.20%Preservative pH 4.0-8.0 Water QS to 100.0 mL

Formulation IX RGCysteic-peptide 0.0001 mg to 10 g Hyaluronic AcidSodium salt  0.01 to 10% Sodium Alginate  0.01 t0 10% Boric Acid  0.01to 1.0% Polyethyleneglycol (PEG 8000)  0.01 to 10% Sodium Chloride  0.10to 0.9% Potassium Chloride  0.01 to 0.20% Calcium Chloride Dihydrate0.001 to 0.05% Magnesium Chloride Hexahydrate  0.01 to 0.20%Preservative pH 3.0-8.0 Water QS to 100.0 mL

Formulation X RGCysteic-peptide 0.0001 mg to 10 g Hyaluronic Acid Sodiumsalt  0.01 to 10% Alginic Acid  0.01 t0 10% Boric Acid  0.01 to 1.0%Polyethyleneglycol (PEG 8000)  0.01 to 10% Sodium Chloride  0.10 to 0.9%Potassium Chloride  0.01 to 0.20% Calcium Chloride Dihydrate 0.001 to0.05% Magnesium Chloride Hexahydrate  0.01 to 0.20% Preservative pH3.0-8.0 Water QS to 100.0 mL

Example 2 Comparison of the PVD-Inducing Effects of RGD Peptides andGlycyl-Arginyl-Glycyl-Cysteic-Threonyl-Proline-COOH (GRG Cysteic AcidTP: Compound 1) in Rabbits

In this example, the PVD-inducing Effects of RGD Peptides andGlycyl-Arginyl-Glycyl-Cysteic-Threonyl-Proline-COOH (RGCysteic AcidPeptide; GRG Cysteic Acid TP; Compound 1) were compared in rabbits. Theprotocol for this study was as follows:

Protocol:

Animal Model

-   -   a) 20 Male and Female Rabbits    -   b) Weighing approximately 1.5-2.5 kg.    -   c) Divide into 2 groups    -   i) 10 Rabbits were injected intravitreally with 2.5% RGD        solution at pH=6.5        -   a) 10 Right Eye injected with 2.5% RGD solution        -   b) 5 Left Eye used as BSS Control        -   c) 5 Left Eyes injected with 2.5% RGD+0.02% EDTA at pH=6.5    -   ii) 10 Rabbits were injected intravitreally with 2.5% RGCysteic        solution at pH=6.5        -   a) 10 Right Eye injected with 2.5% RGCysteic solution at            pH=6.5        -   b) 10 Left Eye injected with 2.5% RGCysteic solution+0.02%            EDTA at pH=6.5

Active Chemicals

-   -   d) Sodium EDTA−99.0-100.5% from Spectrum Chemical Corp.    -   e) RGCysteic acid—cGMP supplier (Purity>98%).    -   f) RGD—cGMP supplier (Purity>98%).    -   g) BSS solution

Both the RGCysteic acid, RGD, RGCysteic acid+Sodium EDTA, RGD+SodiumEDTA, and the BSS solutions were injected into the vitreous cavity 24hours prior to surgery. The rabbits (10 mg/kg body weight) wereanesthetized with an intramuscular injection of 2.0 ml of 1:1combination of xylazine (100 mg/ml) and Ketamine hydrochloride (100mg/ml). Pupils are dilated with topical cyclopentolate hydrochloride 1%and Phenylephrine hydrochloride 10%.

All animals were initially examined with slit lamp biomicroscopy andindirect ophthalmoscopy to exclude any animals with pre-existingvitreoretinal abnormalities. The intravitreal injection of 0.10 cc wasadministered 2 mm posterior to the limbus in the supranasal quadrantusing a 30-gauge needle attached to a 1.0 cc syringe. Care must be takento avoid damage to the lens or retina.

Twenty four hours following injection and immediately before initiationof a mechanical vitrectomy, a B-scan ultrasonography was performed todetermine the status of the posterior vitreous and also the liquefactionof the vitreous. A two-port pars plana vitrectomy was performed using aninfusion fiberoptic and a vitreous cutter attached to a vitrectomy unit.Following a 30 second core vitrectomy, the vitreous cutter was directedto the peripapillary retinal surface where, using low aspiration (<30mmHg) a separation of the posterior cortical vitreous from the retinalsurface was attempted in 4 quadrants. The sclerotomies were sutured anda postoperative B-Scan ultrasonography was carried out to determine thepresence and extent of any PVD present in each quadrant. The animalswere euthanized with intracardiac sodium pentobarbital injections, andthe eyes were immediately enucleated.

Classification of Liquefaction of the Vitreous and PVD was gradedfollowing this grading system to evaluate the extent of PVD based on thepostoperative B-Scan ultrasound examination;

-   -   Grade 0. a) No detachment of the posterior Vitreous is observed.        -   b) Vitreous Liquefaction    -   Grade 1. a) Consists of eyes in which the vitreous is detached        in 2 or less quadrants.        -   b) Vitreous Liquefaction    -   Grade 2. a) Consists of eyes in which the vitreous is detached        in 3 or more quadrants, but with remaining focal attachments        along the medullary rays        -   b) Vitreous Liquefaction    -   Grade 3. a) Consists of eyes in which the vitreous is totally        detached from the retinal surface        -   b) Vitreous Liquefaction

All eyes underwent a sharp razor penetration at the superior polesub-adjacent to the pars plana immediately after enucleating to insurerapid penetration of fixative. Care was taken to avoid damage to theadjacent retina and lens. The eyes were immersed in 2% paraformaldehydeplus 2.5% glutaraldehyde for a minimum of 24 hours at 4 degrees Celsius.A unique posterior calotte was removed, dehydrated in methanol, anddried in carbon dioxide to the critical point, sputter-coated in goldand photographed using the scanning electron microscope.

Results:

Injection: 2.5% RGCysteic acid

Group 1. At Baseline all Animals have no PVD in both eyes Eye ExaminedAnimal 1 Animal 2 Animal 3 Animal 4 Animal 5 O D Treated Grade 3-4 QGrade 3-4 Q Grade 2-3 Q Grade 3-4 Q Grade 1-2 Q Vitreous totallyVitreous totally Vitreous Vitreous totally Vitreous Detached Detacheddetached in 3 Detached detached or more Q in 2 or less Q O S ControlGrade 0 Grade 0 Grade 0 Grade 0 Grade 0Injection: 2.5% of RGD

Group 2. At Baseline at Animals have no PVD in both eyes Eye ExaminedAnimal 1 Animal 2 Animal 3 Animal 4 Animal 5 O D Treated Grade 3-4 QGrade 2-3 Q Grade 1-2 Q Grade 3-4 Q Grade 3-4 Q Vitreous totallyVitreous Vitreous Vitreous totally Vitreous Detached detached in 3 ordetached in 2 Detached totally more Q or less Q Detached O S ControlGrade 0 Grade 1-2 Q Grade 3-4 Q Grade 1-2 Q Grade 0 Vitreous VitreousVitreous detached in 2 or totally detached in 2 or less Q Detached lessQInjection: 2.5% RGCysteic acid+0.02% NaEDTA

Group 3. At Baseline all animals have no PVD in both eyes Eye ExaminedAnimal 1 Animal 2 Animal 3 Animal 4 Animal 5 O D Treated Grade 1-2 QGrade 3-4 Q — Grade 3-4 Q Grade 1-2 Q Vitreous Vitreous totally Vitreoustotally Vitreous detached in 2 Detached Detached detached or less Q in 2or less Q O S Control Grade 1-2 Q Grade 0 — Grade 2-3 Q Grade 0 VitreousVitreous detached in 2 detached in 3 or or less Q more QInjection: 2.5% RGD+0.02% NaEDTA

Group 4. At Baseline all animals have no PVD in both eyes Eye ExaminedAnimal 1 Animal 2 Animal 3 Animal 4 Animal 5 O D Treated Grade 3-4 QGrade 0 Grade 3-4 Q Grade 0 Grade 1-2 Q Vitreous totally Vitreoustotally Vitreous Detached Detached detached in 2 or less Q O S ControlGrade 2-3 Q Grade 2-3 Q Grade 0 Grade 0 Grade 0 Vitreous Vitreousdetached in 3 or detached in 3 or more Q more Q

The results of the kinetic study in Example 4 of this study show thatRGD and RGCysteic acid (GRG Cysteic Acid TP; Compound 1) have similarproperties and injection of 2.5% RGCysteic acid intravitreally causescomplete separation of the vitreous from the retina in 24 hours, and inaddition the vitreous of both the RGD and the RGCysteic acid rabbits arecompletely liquefied.

Over all, the activity of RGCysteic acid is equal or slightly betterthan that of RGD in inducing complete PVD in the rabbits and liquefyingthe vitreous. This is possibly due to its stronger competitive bindingability to the binding sites of integrin-extracellular matrixinteractions than RGD. As described elsewhere herein, sulfonic acids arestronger acids than corresponding carboxylic acids. This higher polarityof the sulfonic acid group leads to stronger intermolecular bonding. Forexample, R-G-Cysteic acid, which has a more polarized O—H bond, may formstronger hydrogen bonds than R-G-Aspartic acid, which has a relativelyless polarized O—H bond, with the amide groups and/or side chains of theamino acids in the integrin binding site and/or have strongerinteractions with metal ions complexed in the integrin binding site.

The results also indicate that when these compounds are administeredwith 0.02% Sodium Edetate, the activity of both RGD as well as RGCysteicacid are not altered.

The results also show that there were no adverse effects or adversesafety effects from the intravitreal injection of the RGCysteic acidcompound Compound 1 or the RGD compound.

Example 3 Safety Study of Multiple Injections of RGC Peptide Compound 1(GRG Cysteic Acid TP) in Rabbit Eyes

In this example, multiple injections of RGC Peptide Compound 1 wereadministered to the eyes of 5 Male and 4 Female New Zealand Rabbitsweighing approximately 1.5-2.5 kg. and the eyes were examined asdescribed in the following paragraphs.

The study then protocol was as follows:

-   -   A) Baseline Examinations: At baseline the right and the left        eyes of all 9 rabbits were examined slit lamp biomicroscopy and        indirect ophthalmoscopy to confirm that no animals had        pre-existing vitreoretinal abnormalities. In addition β-scan        ultrasonography as well as ERG scans were performed on the left        and right eyes of all 9 animals to obtain baseline readings.    -   B) Experimental Treatments: All 9 Rabbits then received        intravitreal injections of either RGC solution or saline        (control). The treatment solutions were prepared as follows:        -   RGC Solution: a 2.5 mg/100 μl solution of RGC Compound 1            containing 0.02 mg of disodium EDTA+ 0.80 mg of sodium            chloride and USP sterile Water for injection having a pH            adjusted to 6.5.        -   Saline (control): a USP Isotonic sterile saline solution            having an adjusted pH of 6.5.

The dosing proceeded as follows:

1) The right eye of each of the 9 rabbits was injected intravitreallywith 100 of the 2.5 mg/100 μl RGC Solution (delivering a dose=2.5 mg ofCompound 1)

2) The Left eye of each of the 9 rabbits was injected intravitreallywith 100 μl of the Saline (control).

3) One day after the initial intravitreal injections, the right and theleft eyes of all 9 Rabbits were examined, by slit lamp biomicroscopy andindirect ophthalmoscopy to check if any of the rabbits have any adverseeffects from the injection.

4) On the 7^(th) day after the 1^(st) injections, the right and the lefteyes of all 9 Rabbits were again examined by slit lamp biomicroscopy andindirect ophthalmoscopy to determine if any of the rabbits exhibitedadverse effects from the injection. In addition ERG scan were performedon all right and left eyes of all the animals to determine if there wereany changes from baseline

5) A group of 3 rabbits, numbers 901, 904 and 909, was then randomlyselected and mechanical vitrectomy was performed on the right and lefteyes of those 3 selected animals to determine the status of theposterior vitreous.

6) The 3 randomly selected animals, numbers 901, 904 and 909, wereeuthanized with intracardiac sodium pentobarbital injections, and theeyes were immediately enucleated. All eyes underwent a sharp razorpenetration at the superior pole sub-adjacent to the pars planaimmediately after enucleation to insure rapid penetration of fixative.Care was taken to avoid damage to the adjacent retina and lens. The eyeswere immersed in 2% paraformaldehyde plus 2.5% gluteraldehyde for aminimum of 24 hours at 4 degrees Celsius. A unique posterior calotte wasremoved, dehydrated in methanol, and dried in carbon dioxide to thecritical point, sputter-coated in gold and photographed using thescanning electron microscope. The other samples were subjected toHistopathological examination.

The remaining 6 Rabbits, numbers 902, 903, 905, 906, 907 and 908 wereinjected a second time 7 days post first injection.

1) The right eye of each of the 6 remaining rabbits was again injectedintravitreally with 100 of the 2.5 mg/100 μl RGC Solution (delivering asecond 2.5 mg dose of Compound 1)

2) The left eye of each of the 6 remaining rabbits was again injectedintravitreally with 100 μl of the Saline (control).

3) One day after the 2^(nd) intravitreal injections, the right and theleft eyes of all 6 remaining rabbits were examined by slit lampbiomicroscopy and indirect ophthalmoscopy to check if any of the rabbitshave any adverse effects from the injection.

4) On the 7^(th) day after the 2^(nd) injections, the right and the lefteyes of all 6 reaming rabbits were examined, by slit lamp biomicroscopyand indirect ophthalmoscopy to check for any adverse effects from theinjections. In addition ERG scans of the left and right eyes of all theanimals were run to determine if there were any changes from thebaseline.

5) Three rabbits, numbers 902, 903, and 907, were then randomly selectedfrom the remaining 6 animals and mechanical vitrectomy was performed onthe right and left eyes of those 3 randomly selected animals todetermine the status of the posterior vitreous.

6) The 3 randomly selected animals, numbers 902, 903, and 907 were theneuthanized with intracardiac sodium pentobarbital injections, and theeyes were immediately enucleated. All eyes underwent a sharp razorpenetration at the superior pole sub-adjacent to the pars planaimmediately after enucleation to insure rapid penetration of fixative.Care was taken to avoid damage to the adjacent retina and lens. The eyeswere immersed in 2% paraformaldehyde plus 2.5% gluteraldehyde for aminimum of 24 hours at 4 degrees Celsius. A unique posterior calotte wasremoved, dehydrated in methanol, and dried in carbon dioxide to thecritical point, sputter-coated in gold and photographed using thescanning electron microscope. The other samples were subjected toHistopathological examination.

The remaining group of 3 Rabbits, numbers 905, 906, and 908, were theninjected a third time, 14 days after the first injection, as follows:

1) The right eye of each of the 3 remaining rabbits was again injectedintravitreally with 100 of the 2.5 mg/100 μl RGC Solution (delivering athird 2.5 mg dose of Compound 1)

2) The left eye of each of the 3 remaining rabbits was again injectedintravitreally with 100 μl of the Saline (control).

3) One day after the 3^(rd) intravitreal injections, the right and theleft eyes of all 6 remaining rabbits were examined, by slit lamp,biomicroscopy and indirect ophthalmoscopy to check if any of the rabbitshave any adverse effects from the injection.

4) On the 7^(th) day after the 3^(rd) injections, the right and the lefteyes of all 3 remaining rabbits were again examined by slit lampbiomicroscopy and indirect ophthalmoscopy to check for any adverseeffects from the injections. In addition ERG scans of the left and righteyes of all the animals were run to determine if there were any changesfrom the baseline.

5) Mechanical vitrectomy was performed on the right and left eyes of the3 remaining animals (Nos. 905, 906 and 908) to determine the status ofthe posterior vitreous.

6) The 3 remaining animals (Nos. 905, 906, and 908) were euthanized withintracardiac sodium pentobarbital injections, and the eyes wereimmediately enucleated. All eyes underwent a sharp razor penetration atthe superior pole sub-adjacent to the pars plana immediately afterenucleation to insure rapid penetration of fixative. Care was taken toavoid damage to the adjacent retina and lens. The eyes were immersed in2% paraformaldehyde plus 2.5% gluteraldehyde for a minimum of 24 hoursat 4 degrees Celsius. A unique posterior calotte was removed, dehydratedin methanol, and dried in carbon dioxide to the critical point,sputter-coated in gold and photographed using the scanning electronmicroscope. The other samples were subjected to Histopathologicalexamination.

3) Active Chemicals

The active chemicals used in this study were as follows:

a. Disodium EDTA−99.0-100.5% from Spectrum Chemical Corp.

b. RGC Peptide (Compound 1)

c. USP sterile isotonic saline solution

4) Study Formulations

a) RGC Solution: 2.5 mg/100 μl solution of RGC containing 0.02 mg ofdisodium EDTA+0.80 mg of sodium chloride and USP sterile water forinjection adjusted to a pH=6.5. Sterile filter through a 0.22μ filterinto a 2.0 mL vial.

b) Saline (Control): USP Isotonic sterile saline solution pH adjusted6.5. Sterile filtered through a 0.22μ filter into a sterile vial.

5) Anesthesia for injection preparation

a. Intramuscular injection of 2.0 mL of a 1:1 combination of xylazine(100 mg/ml) and Ketamine hydrochloride (100 mg/ml)

b. Pupils were dilated with topical cyclopentolate hydrochloride 1% andPhenylephrine hydrochloride 10%

6) Intravitreal Injection Preparation:

A sterile vial containing the RGC solution containing 2.5 mg/100 μl andisotonic sterile saline solution, pH adjusted to 6.5 was provided.

Prior to the injection, the investigator confirmed that there was 0.10cc (100 micro liters) of solution in the 1.0 cc syringe.

7) Injection Procedure:

Since the intravitreal injections do not result in a level of visualdisability sufficient to disrupt the normal daily activity of therabbits, this is not considered a major survival procedure according tothe animal resolution of the Association for Research in Vision andOphthalmology guidelines.

Both the RGC solution as well as the sterile saline solutions wasinjected into the vitreous cavity after the baseline examination of slitlamp Biomicroscopy, Ophthalmoscopy and ERG was completed on the Rabbits.The Rabbits (10 mg/kg body weight) were anesthetized with anintramuscular injection of 2.0 ml of 1:1 combination of xylazine (100mg/ml) and Ketamine hydrochloride (100 mg/ml). Pupils were dilated withtopical cyclopentolate hydrochloride 1% and Phenylephrine hydrochloride10%.

All animals were initially examined with slit lamp biomicroscopy andindirect opthalmoscopy to exclude any animals with pre-existingvitreoretinal abnormalities. The intravitreal injection of 0.10 cc wasadministered 2 mm posterior to the limbus in the supronasal quadrantusing a 30-gauge needle attached to a 1.0 cc syringe. Care was taken toavoid damage to the lens or retina.

Seven (7) days following injection a mechanical Vitrectomy was performedon the animals. A two-port pars plana vitrectomy was performed using aninfusion fiberoptic and a vitreous cutter attached to a vitrectomy unit.Following a 30 second core vitrectomy, the vitreous cutter was directedto the peripapillary retinal surface where, using low aspiration (<30mmHg) a separation of the posterior cortical vitreous from the retinalsurface was attempted in 4 quadrants. The animals were euthanized withintracardiac sodium pentobarbital injections, and the eyes wereimmediately enucleated.

All eyes underwent a sharp razor penetration at the superior polesub-adjacent to the pars plana immediately after enucleation to insurerapid penetration of fixative. Care was taken to avoid damage to theadjacent retina and lens. The eyes were immersed in 2% paraformaldehydeplus 2.5% gluteraldehyde for a minimum of 24 hours at 4 degrees Celsius.A unique posterior calotte was removed, dehydrated in methanol, anddried in carbon dioxide to the critical point, sputter-coated in goldand photographed using the scanning electron microscope.

Analysis of Results

The data on the Safety between eyes treated with RGC solution andsterile saline solution was analyzed for safety using the followingtechniques:

i) Slit lamp biomicroscopy;

ii) Ophthalmoscopy;

iii) ERG;

iv) Histopathology; and

v) Electron microscopy.

Safety Profile:

First Intravitreal administration of 100 μl of 2.5% RGC solution to thegroup of nine Rabbits numbers 901, 902, 903, 904, 905, 906, 907, 908,909, was not associated with any significant toxicity at all timepoints. There was no significant difference in reported adverse effectsbetween the 2.5% RGC group and the isotonic saline solution group. Thislack of toxicity was determined by clinical examination, indirectophthalmoscopy and ultrasound β-scan and mechanical vitrectomy.

Slit lamp biomicroscopy was performed at all study visits and focused onthe lids, conjunctiva and sclera, comea, endothelial changes, anteriorchamber reaction, iris, lens and capsule, as well as the anteriorvitreous for signs for inflammation demonstrate a near complete lack ofinflammatory reaction to intravitreal injections of 100 μl of 2.5% RGCsolution and the isotonic saline solution group. At all study points andin all study groups there did not appear to be any signs of significanttoxicity induced by the test articles.

Clinical evaluation of the posterior segment was also followedthroughout the study to ensure that there was no significant retinaltoxicity present. Indirect ophthalmoscopy as well as slit lamp fundusevaluations were carried out at each evaluation time point with specificattention to any signs of retinal toxicity. The posterior segment wasevaluated for any changes in vitreous density, vitreous liquefaction,vitreous attachment, and possible hemorrhage. The retina was evaluatedfor any signs of RPE toxicity, retinal vascular compromise retinalhemorrhage, exudates, retinal tears, breaks, or detachments. There wereno RPE changes at baseline prior to treatment, at all study points andin all study groups there did not appear to be any signs of significantposterior segment changes induced by the test articles. It is importantto note that ERG scans performed on the nine animals prior to theintravitreal injections, as well as ERG scans performed on all theanimals 1 day and 7 days post injection did not appear to cause anysigns of significant change or toxicity induced by the test articles.

The second Intravitreal administration of 100 μl of 2.5% RGC solution tothe group of six Rabbits numbers 902, 903, 905, 906, 907, 908, was notassociated with any significant toxicity at all time points. There wasno significant difference in reported adverse effects between the 2.5%RGC group and the isotonic saline solution group. This lack of toxicitywas determined by clinical examination, indirect ophthalmoscopy andultrasound β-scan and mechanical vitrectomy.

Slit lamp biomicroscopy was performed at all study visits and focused onthe lids, conjunctiva and sclera, cornea, endothelial changes, anteriorchamber reaction, iris, lens and capsule, as well as the anteriorvitreous for signs for inflammation demonstrate a near complete lack ofinflammatory reaction to intravitreal injections of 100 μl of 2.5% RGCsolution and the isotonic saline solution group. At all study points andin all study groups there did not appear to be any signs of significanttoxicity induced by the test articles.

Clinical evaluation of the posterior segment was also followedthroughout the study to ensure that there was no significant retinaltoxicity present. Indirect ophthalmoscopy as well as slit lamp fundusevaluations were carried out at each evaluation time point with specificattention to any signs of retinal toxicity. The posterior segment wasevaluated for any changes in vitreous density, vitreous liquefaction,vitreous attachment, and possible hemorrhage. The retina was evaluatedfor any signs of RPE toxicity, retinal vascular compromise retinalhemorrhage, exudates, retinal tears, breaks, or detachments. There wereno RPE changes at baseline 7 days prior to the second treatment, at allstudy points and in all study groups there did not appear to be anysigns of significant posterior segment changes induced by the testarticles. It is important to note that ERG scans performed on the sixanimals for a second time post the intravitreal injections, as well asERG scans performed on all the animals 8 days and 14 days post injectiondid not appear to cause any signs of significant change or toxicityinduced by the test articles.

The third Intravitreal administration of 100 μl of 2.5% RGC solution tothe group of six Rabbits numbers 905, 906, 908, was not associated withany significant toxicity at all time points. There was no significantdifference in reported adverse effects between the 2.5% RGC group andthe isotonic saline solution group. This lack of toxicity was determinedby clinical examination, indirect ophthalmoscopy and ultrasound β-scanand mechanical vitrectomy.

Slit lamp biomicroscopy was performed at all study visits and focused onthe lids, conjunctiva and sclera, comea, endothelial changes, anteriorchamber reaction, iris, lens and capsule, as well as the anteriorvitreous for signs for inflammation demonstrate a near complete lack ofinflammatory reaction to intravitreal injections of 100 μl of 2.5% RGCsolution and the isotonic saline solution group. At all study points andin all study groups there did not appear to be any signs of significanttoxicity induced by the test articles.

Clinical evaluation of the posterior segment was also followedthroughout the study to ensure that there was no significant retinaltoxicity present. Indirect ophthalmoscopy as well as slit lamp fundusevaluations were carried out at each evaluation time point with specificattention to any signs of retinal toxicity. The posterior segment wasevaluated for any changes in vitreous density, vitreous liquefaction,vitreous attachment, and possible hemorrhage. The retina was evaluatedfor any signs of RPE toxicity, retinal vascular compromise retinalhemorrhage, exudates, retinal tears, breaks, or detachments. There wereno RPE changes at baseline 14 days prior to the third treatment, at allstudy points and in all study groups there did not appear to be anysigns of significant posterior segment changes induced by the testarticles. It is important to note that ERG scans performed on the threeanimals for a third time post the intravitreal injections, as well asERG scans performed on all the animal 15 days and 21 days post injectiondid not appear to cause any signs of significant change or toxicityinduced by the test articles.

Example 4 Anti-Adhesive Properties of RGC Peptides: Kinetic Study ofWound Healing with Compound 1 (GRG Cysteic Acid TP), Cyclic-RGD and RGE

In this example, in a model of wound healing, it has been demonstratedthat RGC peptides have anti-adhesive properties and therefore canprevent development of many pathological vitreoretinal diseases and caninhibit metastases in human melanoma and colon cancer cells.

To test the anti-adhesive properties of RGC peptides in vitro, awound-healing assay was performed with human umbilical vein endothelialcells (HUVEC). HUVEC were seeded and allowed to grow to a confluentmonolayer on a fibronectin coated surface. A wound (a scratch rift) wascreated by dragging a small pipette tip across the HUVEC monolayer. Thecells were then incubated in fresh growth medium containing the RGCpeptide (Compound 1; 10 mM), and the wound area was imaged in fivedifferent fields at various time points (0, 4, 8, 12, 16, 20, 24 hours)to determine the kinetics of wound closure. The level of wound closurewas quantified by determining the fraction of the original wound areathat was re-occupied by HUVEC through cellular adhesion, migration andproliferation.

In control studies, the following peptides were used in place of RGC(Compound 1): cyclic-RGD peptide (1 mM), positive control) and RGEpeptide (1 mM, negative control).

The effect of the peptides on the kinetics of wound healing is presentedin FIG. 1.

The results are shown as a percentage of the original area. The errorbars correspond to the standard deviation in wound size across 2-6independent trials.

The results of the kinetics of HUVEC wound closure show that the RGCpeptide inhibits HUVEC wound healing by 70% after 24 hours, while CyclicRGD (RGD-based peptide; N-methylated cyclic-RGDf-N(Me)V; Cilengitide)inhibits HUVEC wound healing by 45% after 24 hours. These were bothcompared to a negative control RGE peptide that inhibited HUVEC woundhealing by 0% after 24 hours. The effect of RGC (Compound 1) isquantitatively comparable to the activity of the RGD-based peptide, awell established inhibitor of integrin binding activity. Further, RGCpeptide exhibits similar properties to the activities of the RGD-basedpeptide without apoptosis of the HUVEC cells.

As described elsewhere herein, the strong adhesion between the vitreousand the retina could account for eventual development of manypathological vitreoretinal diseases such as vitreomacular traction,proliferative diabetic retinopathy, macular hole, age related maculardegeneration and floaters. Thus, an atraumatic non-invasive approach toachieve a Posterior Vitreous Detachment, other than the mechanicalseparation of the vitreous from the inner retinal surface, is highlydesirable (see Tezel, T. H. et al, Retina (1998) 18: 7-15; andVerstraeten, T. C, et al., Arch. Ophthalmol. (1993)111: 849-854).

As described elsewhere herein, it is believed that the ECM components,particularly collagen fibrils of the cortical vitreous, are anchored tothe inner surface of the retina through integrin binding sites (seeFoos, R. Y., Invest. Ophthalmol. Vis. Sci. (1972) 11:801-808) in theinner limiting lamella (ILL). It is also known that major adhesiveglycoproteins of the ILL in the eye such as fibronectin and laminin, areheavily linked to integrins (see Curtis, T. M. et al., Am. J. Physiol.,(1995) 269: L248-L260; Elner, S. G., et al., IOVS (1996) 37:696-701; andHorman, S. M, et al., Am. J. Physiol. (1995) 269: L248-L260) through theRGD (Arg-Gly-Asp) sequences and several integrins bind through RGD motifpresent in ECM proteins. Further, it is known that fibronectin binds toseveral other integrins besides α_(y)β₃, whereas vitronectin isα_(v)β₃-specific.

The primary connection of the integrins to the ECM involves theArg-Gly-Asp(RGD)sequence and the RGD sequence binds to a shallow crevicelocated between the α- and β-subunits of the integrin head (see Xiong,et al., Science (2002) 296: 151-155). Such binding helps modulatevarious cellular signaling pathways, including cell adhesion, migration,differentiation, angiogenesis and wound healing (see Ruoslahti, E., etal., Science (1987) 238: 491-497; and J. Clin. Invest. (1991) 87: 1-5).

Since the vitreous extracellular matrix; e.g., collagen fibrils areconnected to the cellular retina by the integrin binding sites,intravitreal injection of RGC peptides (oligopeptides) could release theRGD motif of the vitreous extracellular matrix from the cellular retinaby a competitive binding to the same integrin receptor sites.

Several investigators (see Ruoslahti, E. et al., Science (1987) 238:491-497; Hynes, R. A., et al., Cell (1992) 68: 303-322; and Humphries,M. J., J. Cell Sci., (1990) 97: 585-592) demonstrated that manyintegrins (α_(v)β₃, α₅β₁, α₁₁β₃, etc.) can be inhibited by smallpeptides that possess the RGD sequence motif. It is also well documentedthat that α_(v)β₃ and α₅β₁ integrins, as well as vitronectin andfibronectin, were upregulated in tumors such as human melanoma cells(see Nip, J., J. Clin. Invest., (1992) 90: 1406-1413), human breastcancer cells (see Rong, L. et al., Invest. Ophthalmol. Vis. Sci. (2009)50: 5988-5996), and human retinal pigment epithelial cells (Peter C.Brooks, et al., J. Clin. Invest., (1995) 96: 1815-1822). Thus it hasbeen demonstrated that there is good correlation between metastaticpotentials of human melanoma cells and adhesion of melanoma cells tolymph node vitronectin via the α_(v)β₃ integrin receptor and that theadhesion was inhibited by an RGD containing peptide (Nip, J., J. Clin.Invest., (1992) 90: 1406-1413). This demonstrates that RGD peptides canbe an important anti-angiogenic agents.

Further, it has been demonstrated in a human colon cancer cell line thatwhen there is significant increase in cell adhesion, then there isincreased metastatic activity (Lehmann, M., Cancer Res., (1994), 54:2102-2107). Therefore agents that inhibit cell adhesion effectivelyinhibit colon cancer and melanoma from metastasizing.

Based on the results of the wound healing study where RGC has been shownto inhibit cell adhesion, and upon extrapolation of the metastaticpotential of RGD in the Melanoma and Colon cancer models, RGC peptidesand their derivatives can effectively inhibit tumor metastases, forexample, in melanoma and colon cancer.

Example 5 Use of RGC Peptides for Directing or Delivering Agents toTumors

In this example, a dimeric RGC Peptide-Paclitaxel conjugate (Compound3), shown below, is provided. This composition is useful as an antitumoragent. The dimeric RGC Peptide selectively binds to integrin receptorsthat are highly expressed in certain cancer cells and is useful to treatcertain metastatic cancers such as, for example, metastatic breastcancer, by inhibiting cell adhesion.

The synthesis and mechanisms of action, biodistribution and tumorselectivity of the corresponding RGD analogue of Compound 3 are asdescribed in Chen, X., et al.; Synthesis and Biological Evaluation ofDimeric RGD Peptide-Paclitaxel Conjugate as a Model forIntegrin-Targeted Drug Delivery; J. Med. Chem., (2005) 48 (4):1098-1106).

Although Compound 3 comprises a particular anti-tumor agent, Paclitaxel,bound to dimeric RGC peptide, it is to be appreciated that this aspectof the invention includes all monomeric or multimeric forms of RGCpeptides bound to any feasible diagnostic or therapeutic agents that maybe useful in diagnosing, imaging or treating a tumor or otherintegrin-containing tissue or structure. Examples of antitumorsubstances that may be bound to monomeric or multimeric RGC peptides inaccordance with this invention may include antitumor agents (e.g.,cancer chemotherapeutic agents, biological response modifiers,vascularization inhibitors, hormone receptor blockers, cryotherapeuticagents or other agents that destroy or inhibit neoplasia ortumorigenesis) such as; alkylating agents or other agents which directlykill cancer cells by attacking their DNA (e.g., cyclophosphamide,isophosphamide), nitrosoureas or other agents which kill cancer cells byinhibiting changes necessary for cellular DNA repair (e.g., carmustine(BCNU) and lomustine (CCNU)), antimetabolites and other agents thatblock cancer cell growth by interfering with certain cell functions,usually DNA synthesis (e.g., 6 mercaptopurine and 5-fluorouracil (5FU),antitumor antibiotics and other compounds that act by binding orintercalating DNA and preventing RNA synthesis (e.g., doxorubicin,daunorubicin, epirubicin, idarubicin, mitomycin-C and bleomycin) plant(vinca) alkaloids and other anti-tumor agents derived from plants (e.g.,vincristine and vinblastine), steroid hormones, hormone inhibitors,hormone receptor antagonists and other agents which affect the growth ofhormone-responsive cancers (e.g., tamoxifen, herceptin, aromataseingibitors such as aminoglutethamide and formestane, triazole inhibitorssuch as letrozole and anastrozole, steroidal inhibitors such asexemestane), anti-angiogenic proteins, small molecules, gene therapiesand/or other agents that inhibit angiogenesis or vascularization oftumors (e.g., meth-1, meth-2, thalidomide), bevacizumab (Avastin),squalamine, endostatin, angiostatin, Angiozyme, AE-941 (Neovastat),CC-5013 (Revimid), medi-522 (Vitaxin), 2-methoxyestradiol (2ME2,Panzem), carboxyamidotriazole (CAI), combretastatin A4 prodrug (CA4P),SU6668, SU11248, BMS-275291, COL-3, EMD 121974, IMC-1C11, IM862,TNP-470, celecoxib (Celebrex), rofecoxib (Vioxx), interferon alpha,interleukin-12 (IL-12) or any of the compounds identified in ScienceVol. 289, Pages 1197-1201 (Aug. 17, 2000) which is expresslyincorporated herein by reference, biological response modifiers (e.g.,interferon, bacillus calmette-guerin (BCG), monoclonal antibodies,interluken 2, granulocyte colony stimulating factor (GCSF), etc.), PGDFreceptor antagonists, herceptin, asparaginase, busulphan, carboplatin,cisplatin, carmustine, chlorambucil, cytarabine, dacarbazine, etoposide,flucarbazone, flurouracil, gemcitabine, hydroxyurea, ifosphamide,irinotecan, lomustine, melphalan, mercaptopurine, methotrexate,thioguanine, thiotepa, tomudex, topotecan, treosulfan, vinblastine,vincristine, mitoazitrone, oxaliplatin, procarbazine, streptocin, taxol,taxotere, analogs/congeners and derivatives of such compounds as well asother antitumor agents not listed here.

Example 6 ⁶⁴CU-Labeled Multimeric RGC Peptides for Imaging ofIntegrin-Expressive Tumors

In this example, ⁶⁴CU-Labeled tetrameric and octameric RGC peptides ofthe present invention (Compounds 4 and 5 respectively), shown below, areuseful as radiotherapeutic agents for imaging and diagnostic purposes(e.g., radiolabeling of tumors for PET scanning) as well as fordirecting or delivering therapeutic agents to tumors or other cellswhich express integrins, such as tumors which express α_(v)β₃ integrins.

The synthesis and mechanisms of action, biodistribution, tumorselectivity and PET related use of the corresponding RGD analogues ofCompounds 4 and 5 are as described in Li, Z. et al., ⁶⁴CU-LabeledTetrameric and Octameric RGD Peptides for Small-Animal PET of Tumorα_(v)β₃ Integrin Expression; J. Nucl. Med. 48 (7) pp. 1162-1171 (2007).

As used herein, any reference to treating or treatment of a disease ordisorder shall be construed to include preventing or prevention of thedisease or disorder before it has occurred or been detected as well astreating the disease or disorder after it has occurred or has beendetected.

It is to be appreciated that the invention has been described here abovewith reference to certain examples or embodiments of the invention butthat various additions, deletions, alterations and modifications may bemade to those examples and embodiments without departing from theintended spirit and scope of the invention. For example, any element orattribute of one embodiment or example may be incorporated into or usedwith another embodiment or example, unless otherwise specified of if todo so would render the embodiment or example unsuitable for its intendeduse. Also, where the steps of a method or process have been described orlisted in a particular order, the order of such steps may be changedunless otherwise specified or unless doing so would render the method orprocess unworkable for its intended purpose. All reasonable additions,deletions, modifications and alterations are to be consideredequivalents of the described examples and embodiments and are to beincluded within the scope of the following claims. All publications andpatent documents cited herein are hereby incorporated by reference intheir entirety for all purposes to the same extent as if each were soindividually denoted.

What is claimed is:
 1. A method of inhibiting adhesion of cells to RGDbinding sites comprising administering to a subject in need thereof aneffective amount of a cyclic or linear compound comprising either: i) apeptide which comprisesGlycinyl-Arginyl-Glycinyl-Cysteic-Threonyl-Proline-COOH or ii) a peptidewhich has the formula:X₁—R-G-Cysteic Acid-X where X and X₁ are selected from: Phe-Val-Ala,-Phe-Leu-Ala, -Phe-Val-Gly, -Phe-Leu-Gly, -Phe-Pro-Gly, -Phe-Pro-Ala,-Phe-Val; or from Arg, Gly, Cysteic, Phe, Val, Ala, Leu, Pro, Thr andsalts thereof and any combinations of D-isomers and L-isomers thereof.2. The method of claim 1 wherein the peptide is administered to thesubject's eye and wherein said inhibition of adhesion of cells to RGDbinding sites is effective to treat a disorder of the subject's eye. 3.The method of claim 2 wherein the peptide is administered byintravitreal injection.
 4. The method of claim 2 wherein said inhibitionof adhesion of cells to RGD binding sites is effective to treat adisorder of the subject's eye selected from: neovascularization,neovascular glaucoma, diabetic retinopa thy, retinal degeneration, drymacular degeneration, wet macular degeneration, cornealneovascularization and vitreo-macular traction.
 5. The method of claim 1wherein the peptide is administered systemically and wherein saidinhibition of adhesion of cells to RGD binding sites is effective totreat a cancer which expresses an integrin that recognizes the RGDmotif.
 6. The method of claim 5 wherein the cancer comprises avascularized solid tumor or metastatic lesions from a vascularized solidtumor.
 7. The method of claim 1 wherein the peptide has the structuralformula:


8. The method of claim 1 wherein the peptide has the structural formula:

where X′ is selected from: H, C1-C₆ alkyl, Ph or SO₃H and Z is selectedfrom H or Me; Y is selected from OH, NH₂.
 9. The method of claim 1wherein the peptide comprises Glycine-Arginine-Glycine-Cysteic(Acid)-Threonine-Proline.
 10. A method of antagonizing an RGD-bindingintegrin in a subject comprising administering to a subject in needthereof an effective amount of a cyclic or linear compound comprisingeither: i) a peptide which comprisesGlycinyl-Arginyl-Glycinyl-Cysteic-Threonyl-Proline-COOH or ii) a peptidewhich has the formula:X₁—R-G-Cysteic Acid-X where X and X₁ are selected from: Phe-Val-Ala,-Phe-Leu-Ala, -Phe-Val-Gly, -Phe-Leu-Gly, -Phe-Pro-Gly, -Phe-Pro-Ala,-Phe-Val; or from Arg, Gly, Cysteic, Phe, Val, Ala, Leu, Pro, Thr andsalts thereof and any combinations of D-isomers and L-isomers thereof.11. The method of claim 10 wherein the peptide is administered to thesubject's eye and wherein said antagonizing of an RGD-binding integrinis effective to treat a disorder of the subject's eye.
 12. The method ofclaim 11 wherein the peptide is administered by intravitreal injection.13. The method of claim 11 wherein said antagonizing of an RGD-bindingintegrin is effective to treat a disorder of the subject's eye selectedfrom: neovascularization, neovascular glaucoma, diabetic retinopathy,retinal degeneration, dry macular degeneration, wet maculardegeneration, corneal neovascularization and vitreo-macular traction.14. The method of claim 10 wherein said antagonizing of an RGD-bindingintegrin is effective to treat a cancer which expresses an integrin thatrecognizes the RGD motif.
 15. The method of claim 14 wherein the cancercomprises a vascularized solid tumor or metastatic lesions from avascularized solid tumor.
 16. The method of claim 10 wherein the peptidehas the structural formula:


17. The method of claim 10 wherein the peptide has the structuralformula:

where X′ is selected from: H, C1-C₆ alkyl, Ph or SO₃H and Z is selectedfrom H or Me; Y is selected from OH, NH₂.
 18. The method of claim 10wherein the peptide comprises Glycine-Arginine-Glycine-Cysteic(Acid)-Threonine-Proline.
 19. A method of antagonizing an integrin in asubject comprising administering to a subject in need thereof a cyclicor linear compound comprising either: i) a peptide which comprisesGlycinyl-Arginyl-Glycinyl-Cysteic-Threonyl-Proline-COOH or ii) a peptidewhich has the formula:X₁—R-G-Cysteic Acid-X where X and X₁ are selected from: Phe-Val-Ala,-Phe-Leu-Ala, -Phe-Val-Gly, -Phe-Leu-Gly, -Phe-Pro-Gly, -Phe-Pro-Ala,-Phe-Val; or from Arg, Gly, Cysteic, Phe, Val, Ala, Leu, Pro, Thr andsalts thereof and any combinations of D-isomers and L-isomers thereof,wherein said integrin is one which is subject to antagonism by an RGDpeptide.
 20. The method of claim 19 wherein the peptide is administeredto the subject's eye and said antagonizing of an integrin is effectiveto treat a disorder of the subject's eye.
 21. The method of claim 20wherein the peptide is administered by intravitreal injection.
 22. Themethod of claim 20 wherein the antagonizing of an integrin is effectiveto treat a disorder of the subject's eye selected from:neovascularization, neovascular glaucoma, diabetic retinopathy, retinaldegeneration, dry macular degeneration, wet macular degeneration,corneal neovascularization and vitreo-macular traction.
 23. The methodof claim 19 wherein the peptide is administered systemically to treat acancer which expresses an integrin that recognizes the RGD motif. 24.The method of claim 23 wherein the cancer comprises a vascularized solidtumor or metastatic lesions from a vascularized solid tumor.
 25. Themethod of claim 19 wherein the peptide has the structural formula:


26. The method of claim 19 wherein the peptide has the structuralformula:

where X′ is selected from: H, C1-C₆ alkyl, Ph or SO₃H and Z is selectedfrom H or Me; Y is selected from OH or NH₂.
 27. The method of claim 19wherein the peptide comprises Glycine-Arginine-Glycine-Cysteic(Acid)-Threonine-Pro.